WO2013098294A1 - Procédé de mesure du monoxyde d'azote dans le sang - Google Patents

Procédé de mesure du monoxyde d'azote dans le sang Download PDF

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WO2013098294A1
WO2013098294A1 PCT/EP2012/076904 EP2012076904W WO2013098294A1 WO 2013098294 A1 WO2013098294 A1 WO 2013098294A1 EP 2012076904 W EP2012076904 W EP 2012076904W WO 2013098294 A1 WO2013098294 A1 WO 2013098294A1
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blood
subsample
epr
spectrum
rbcs
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PCT/EP2012/076904
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English (en)
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Jean-Luc Balligand
Irina LOBYSHEVA
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L'université Catholique De Louvain
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Priority to US14/367,209 priority Critical patent/US9696324B2/en
Priority to EP12808418.3A priority patent/EP2798365A1/fr
Publication of WO2013098294A1 publication Critical patent/WO2013098294A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/72—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood pigments, e.g. haemoglobin, bilirubin or other porphyrins; involving occult blood
    • G01N33/721—Haemoglobin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01R—MEASURING ELECTRIC VARIABLES; MEASURING MAGNETIC VARIABLES
    • G01R33/00—Arrangements or instruments for measuring magnetic variables
    • G01R33/20—Arrangements or instruments for measuring magnetic variables involving magnetic resonance
    • G01R33/60—Arrangements or instruments for measuring magnetic variables involving magnetic resonance using electron paramagnetic resonance
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/15—Devices for taking samples of blood
    • A61B5/150007—Details
    • A61B5/150015—Source of blood
    • A61B5/15003—Source of blood for venous or arterial blood
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/15—Devices for taking samples of blood
    • A61B5/150007—Details
    • A61B5/150732—Needle holders, for instance for holding the needle by the hub, used for example with double-ended needle and pre-evacuated tube
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/15—Devices for taking samples of blood
    • A61B5/150007—Details
    • A61B5/15074—Needle sets comprising wings, e.g. butterfly type, for ease of handling
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/15—Devices for taking samples of blood
    • A61B5/150007—Details
    • A61B5/150755—Blood sample preparation for further analysis, e.g. by separating blood components or by mixing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/15—Devices for taking samples of blood
    • A61B5/153—Devices specially adapted for taking samples of venous or arterial blood, e.g. with syringes
    • A61B5/154—Devices using pre-evacuated means
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/49—Blood
    • G01N33/4925—Blood measuring blood gas content, e.g. O2, CO2, HCO3
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/84—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving inorganic compounds or pH
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/0004—Gaseous mixtures, e.g. polluted air
    • G01N33/0009—General constructional details of gas analysers, e.g. portable test equipment
    • G01N33/0027—General constructional details of gas analysers, e.g. portable test equipment concerning the detector
    • G01N33/0036—General constructional details of gas analysers, e.g. portable test equipment concerning the detector specially adapted to detect a particular component
    • G01N33/0037—NOx
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/20—Air quality improvement or preservation, e.g. vehicle emission control or emission reduction by using catalytic converters

Definitions

  • the present invention relates to the measurement of nitric oxide (NO) amounts accumulated in blood. More precisely, the present invention relates to the measurement of NO amounts accumulated in vivo in blood using electron paramagnetic resonance (EPR).
  • EPR electron paramagnetic resonance
  • Nitric oxide is a paramagnetic molecule. The detection of NO in biological tissues and liquids is a challenge because of its low concentration and small half-life.
  • a proper assay of the level of bioavailable nitric oxide in vivo in human circulation is important for the protection of human health.
  • Insufficiency of NO production from the endothelium is a crucial sign of endothelial dysfunction in many metabolic diseases and, especially, cardiovascular diseases developed under various risk factors including age, hypertension, smoking, and hypercholesterolemia.
  • the functionality of the endothelial nitric oxide synthase (eNOS) and NO bioavailability in the vascular bed in vivo are difficult to assess quantitatively, especially in humans.
  • NO can be converted into nitrite and nitrate in reactions with various specific and non-specific targets. Therefore, measurements of nitrite/nitrate (NO x ) concentration in extracellular fluids such as blood plasma have been widely used in different laboratories.
  • the Griess reaction detects nitrite based on its reaction with sulphanilic acid. The reaction product is then detected by spectrophotometry.
  • the chemiluminescence technique provides higher sensitivity using back-reduction of nitrite/nitrate to NO in a reflux chamber at 95°C. Both techniques allow nitrite/nitrate detection in biological samples, however, proper data interpretation is difficult due to the dietary variability and the active nitrite/nitrate metabolism.
  • peroxynitrite a strong oxidant produced by the reaction of superoxide anions and NO and detected at high level in pathophysiological models, is also converted to NO x as end product.
  • the level of NO production can be detected a nnpero metrically by NO-specific electrodes.
  • the method requires catheterization in order to insert the electrode close to the endothelium, which is very fragile and can be damaged by this invasive procedure and the application of the method is limited by the sensitivity/specificity of the signal depending on the sensor.
  • EndoPAT is a standardized device for non-invasive endothelial function assessment.
  • the technology is based on the detection of peripheral arterial tone signal using volume-sensitive sensors placed on the fingertips.
  • the EndoPAT method can only be used as an indirect assessment of endothelial NO production.
  • Nitric oxide is known to bind tightly to hemoglobin (Hb). Interactions of NO with Hb are believed to be a major route of NO metabolism in the vascular bed. It follows that the levels of Hb-NO in blood are an excellent indication of endogenous NO production.
  • Distinct forms of paramagnetic heme-NO adducts can be formed depending on NO hosting at a- or ⁇ -subunits and the Hb conformation state under variation of oxygen pressure and allosteric factor abundance in erythrocytes.
  • Electron Paramagnetic Resonance is a method for the individual detection of the level of various paramagnetic compounds in biological samples.
  • EPR is an extremely attractive technique to analyze NO bioavailability. Measurement of paramagnetic hemoglobin-nitric oxide adducts (Hb-NO) in whole blood and erythrocytes by the EPR spectroscopy was proposed in the last decade in animal models and human blood. Unique information about systemic NO levels could be obtained especially in animal models.
  • Hb-NO paramagnetic hemoglobin-nitric oxide adducts
  • the EPR spectra of whole venous human blood showed a number of additional paramagnetic species with wide-line width assigned to Cu-containing proteins, such as ceruloplasmin, and narrow-line width signal with g ⁇ 2 assigned to protein-centered free radicals formed in the erythrocytes.
  • ceruloplasmin and narrow-line width signal with g ⁇ 2 assigned to protein-centered free radicals formed in the erythrocytes.
  • the superposition of the EPR signals from different paramagnetic centers in the same region (g ⁇ 2) where the Hb-NO signal is observed made the Hb-NO quantitation problematic.
  • the background signal of ceruloplasmin could be eliminated by separating the plasma from the erythrocytes while the free radical signal still represents a background signal that interferes with Hb-NO quantitation in vivo in human blood.
  • the present invention aims to provide a solution to at least the above-mentioned problem by providing a method for an accurate NO quantification in vivo in human blood.
  • the present invention provides a method for the quantification of NO in a whole blood sample comprising the steps of collecting a whole blood sample, dividing said collected whole blood sample into at least two blood subsamples, chemically treating at least one blood subsample to at least partly reduce at least one component of said blood subsample, performing EPR measurement of said at least two blood subsamples, performing a first comparison of said chemically treated at least one blood subsample EPR measurement with the EPR measurement of at least one non-chemically treated blood subsample thereby obtaining a first comparison result.
  • the present invention provides a method further comprising the steps of: performing a second comparison wherein said first comparison result is compared with the EPR measurement of at least one non-treated blood subsample thereby obtaining a second comparison result;
  • the present invention provides a method wherein the whole blood sample is collected in an anaerobic way. This is advantageous as it will prevent the dissociation of the in vivo formed Hb-NO complex.
  • the present invention provides a method wherein the red blood cells (RBCs or erythrocytes) of the collected blood sample are separated from the plasma of said collected whole blood sample. This is advantageous as the background signal of ceruloplasmin will be eliminated.
  • the obtained RBCs are divided in at least two blood subsamples.
  • the present invention provides a method wherein chemically treating at least one blood subsample comprises the addition of a chemical compound that will reduce at least partly the free radicals present in the treated blood subsample.
  • the present invention provides a method wherein said chemical compound comprises an antioxidant that is selected from the group of N- acetyl cysteine, a-tocopherol, ascorbic acid and mixtures thereof such as a mixture of ⁇ -tocopherol and ascorbic acid, a mixture of ⁇ -tocopherol and N-acetyl cysteine, preferably said antioxidant is ascorbic acid.
  • said chemical compound consists of at least one antioxidant selected from the group of N-acetyl cysteine, ⁇ -tocopherol, Trolox, a water soluble analog of a-tocopherol, ascorbic acid and mixtures thereof such as a mixture of ⁇ -tocopherol and ascorbic acid, a mixture of ⁇ -tocopherol and N-acetyl cysteine, preferably said antioxidant is ascorbic acid.
  • the added antioxidant has preferably a molar concentration comprised between 1 mM and 20 mM, preferably between 2 mM and 15 mM, more preferably between 3 mM and 12 mM, even more preferably around 10 mM.
  • said at least one chemically treated blood subsample is allowed to settle before the addition of said chemical compound.
  • the present invention provides a method wherein at least one blood subsample is not chemically treated. Said at least one non- chemically treated blood subsample is physically treated by allowing said blood subsample to settle which will result in the dissociation of NO from Hb.
  • the present invention relates to a method wherein at least one blood subsample is not treated. Said at least one blood subsample is immediately frozen after being sampled from said collected whole blood sample.
  • EPR measurements are performed for all blood subsamples.
  • the EPR spectrum of the at least one chemically treated blood subsample is then subtracted from the EPR spectrum of the at least one non-chemically treated blood subsample. This will result in a model EPR spectrum of free radicals specific to the collected whole blood sample.
  • the obtained model spectrum of free radicals is then subtracted from the EPR spectrum of said at least one non-treated blood subsample. This second subtraction will provide an EPR spectrum with eliminated free radicals background and from which the quantitation of Hb-NO is performed using hyperfine component.
  • the present invention provides a method wherein said whole blood sample is a human whole blood sample.
  • the present invention provides a kit suitable for carrying a method of NO measurement, comprising at least a winged infusion set with needle (21Gx3/4"), a blood collection container, gas-tight vacuum tube with adaptor for blood sampling, a chemical solution, at least 3 Pasteur glass or plastic pipettes, at least 3 plastic syringes (1 ml) cut off at the end for subsample freezing, at least 3 small sheets of parafilm to close open end of syringe after filling and a protocol description.
  • the present invention provides a kit wherein said blood collection container is a gas-tight vacuum tube containing an anticoagulant.
  • the present invention provides a kit wherein said chemical product is an antioxidant, preferably ascorbic acid.
  • the present invention provides a method and a kit suitable to be used for the prediction of NO related diseases and the necessary medication treatment.
  • the method, kit and uses provided by the invention are advantageous.
  • the method is a minimally-invasive human approach. It delivers a precise assessment of the Hb-NO level.
  • the invention is very attractive for providing surrogate endpoints in clinical studies for the assessment of medications efficacy or preventive strategies targeting endothelial function in very large populations of patients with risk factors for several diseases such as but not limited to pulmonary hypertension, heart failure with normal ejection fraction (HFNEF), all other forms of heart failure, atherosclerosis, all vascular ischemic diseases, diabetes, high blood pressure, dyslipidemias, metabolic syndrome, obstructive sleep apnea, all systemic inflammatory diseases.
  • the method and kit according to the present invention provide also the possibility for monitoring the side effects of anti-cancer drugs on vascular endothelium, monitoring of blood NO in systemic inflammatory diseases and monitoring the blood NO production in response to exercise.
  • the method according to the invention provides a surrogate end-point in prospective clinical trials. This leads to the reduction of clinical trials costs and the reduction of medications tested for cardiovascular treatments by more precise, fast and adequate individual characterization of vascular system functionality and efficacy of medication. In this way, the method can be helpful in the development of the treatment tailoring and personalization of medicine in future perspectives. DESCRIPTION OF THE FIGURES
  • FIG. 1 Model EPR spectrum of free radicals in RBCs (a) obtained after subtraction of the EPR spectrum (b) of the second subsample of RBCs treated with ascorbic acid (AA) from the EPR spectrum (c) of the third subsample (allowed to settle and not treated with AA).
  • the y-axis represents the intensity in arbitrary units (a.un.).
  • the x- axis represents the magnetic field (Gauss, G) and g is a g-factor of the EPR signal.
  • FIG. 2 Example of double subtraction (volunteer 1).
  • the y-axis represents the intensity in arbitrary units.
  • the x- axis represents the magnetic field, G.
  • FIG. 3 Example of double subtraction (volunteer 2).
  • the y-axis represents the intensity in arbitrary units.
  • the x- axis represents the magnetic field G.
  • FIG. 4 Linear regression analysis between the index of reactive hyperaemia response (RHI) and the Hb-NO level quantified after subtraction of model free radical signal.
  • FIG. 4-1 Linear regression analysis between basal Hb-NO level in RBCs isolated from venous blood, quantified after subtraction of model free radical signal, and the Framingham reactive hyperemia index (FRHI) defined as the natural logarithm of the ratio of mean post-deflation signal (in the 90 to 120-second post-deflation interval) to baseline signal in hyperemic finger normalized by the same ratio in the contra-lateral finger.
  • FRHI Framingham reactive hyperemia index
  • FIG. 5 (A) Calibration curve for quantitation of the Hb-NO level. (B) EPR signals of Hb-NO formed in intact RBCs treated with known concentration of nitrite in presence of Na 2 S 2 0 4 in anaerobic condition.
  • FIG. 6 Individual model EPR spectrum of free radicals (a) in RBCs with prooxidant profile was obtained after inverse subtraction of the EPR spectrum of the third non- treated subsample (c) from the EPR spectrum of second subsample treated with AA (b).
  • FIG. 7 A. Individual model EPR spectrum of free radicals (a) in RBCs obtained after subtraction of the EPR spectrum (b) of the subsample treated with N-acetyl cysteine, from the EPR spectrum of third non-treated subsample (c).
  • FIG. 7-IA Individual model EPR spectra of free radicals (a and a ' ) in RBCs obtained after subtraction of the EPR spectra (b and b ' , correspondingly) of the subsample treated with different N-acetyl cysteine (NAC) concentrations, 5 (b) and 10 mM (b ' ), from the EPR spectrum of third non-treated subsample (c).
  • NAC N-acetyl cysteine
  • FIG. 7-IB Comparative model EPR spectra (a and a ' ) of free radicals obtained as described in FIG. 7-IA, but after treatment of second subsamples b and b' with different concentrations of ascorbic acid, 2 mM (b) and 10 mM (b ' ).
  • FIG. 7-IC Comparison of individual model EPR spectra of free radicals, obtained from RBCs of a single volunteer, are shown after subtraction of the EPR spectrum of the subsample treated with different antioxidants from the EPR spectrum of third non-treated subsample of the same volunteer as described in FIG. 7-IA and FIG. 7-IB. Tested antioxidants were a-tocopherol (a), Trolox (b), a water-soluble analog of ⁇ -tocopherol, and N-acetyl cysteine (c).
  • FIG. 8 Linear regression analysis between the index of reactive hyperaemia response (RHI) and the Hb-NO level, quantified after subtraction of model free radical signal.
  • Fig. 8-1 Linear regression analysis between basal Hb-NO level in RBCs isolated from venous blood, quantified after subtraction of model free radical signal, and the Framingham reactive hyperemia index (FRHI) defined as the natural logarithm of the ratio of mean post-deflation signal (in the 90 to 120-second post-deflation interval) to baseline signal in hyperemic finger normalized by the same ratio in the contra-lateral finger.
  • FRHI Framingham reactive hyperemia index
  • Fig. 9 Quantitative detection of NO formation in circulation, measured as accumulation of Hb-NO in RBCs, obtained from a volunteer after in vivo treatment with sublingual glyceryl trinitrate (GTN).
  • GTN sublingual glyceryl trinitrate
  • Fig. 10 Quantitative detection of NO formation in circulation, measured as the accumulation of Hb-NO in RBCs, obtained from a volunteer after five-minute exercise (stair climbing).
  • Typical EPR spectra of RBCs (i"), frozen after incubation under low 0 2 level with the NO-donor system as described in Fig. 5, are shown at the bottom of the figure for comparison.
  • the present invention relates to the measurement of nitric oxide (NO) amounts accumulated in blood.
  • a compartment refers to one or more than one compartment.
  • “About” as used herein referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, is meant to encompass variations of +/- 20% or less, preferably +/-10% or less, more preferably +/-5% or less, even more preferably +/-1% or less, and still more preferably +/-0.1% or less of and from the specified value, in so far such variations are appropriate to perform in the disclosed invention.
  • the value to which the modifier "about” refers is itself also specifically disclosed.
  • % by weight refers to the relative weight of the respective component based on the overall weight of the formulation.
  • EPR measurement and “EPR spectrum” are used herein as synonyms.
  • whole blood blood from which no constituent, such as red blood cells, white blood cells, plasma, or platelets, has been removed.
  • sample also referred to as “blood subsample” “RBC subsample” or “erythrocytes subsample” is meant the red blood cells obtained from the whole blood sample and divided in at least two different samples.
  • R is a correlation coefficient
  • P-value is the probability that the linear correlation observed may be obtained by chance; it is considered statistically significant at a level ⁇ 0.05
  • N is the number of subjects.
  • the present invention provides a method for the quantification of NO in a whole blood sample comprising the steps of: collecting a whole blood sample; dividing said collected whole blood sample into at least two blood subsamples; chemically treating at least one blood subsample by adding of at least one antioxidant to at least partly reduce free radicals of said blood subsample; performing EPR measurement of said at least two blood subsamples; performing a first comparison which is a subtraction of said chemically treated blood subsample EPR measurement from the EPR measurement of one non-chemically treated blood subsample thereby obtaining a first comparison result which is an EPR spectrum of protein-centered free radicals; performing a second comparison wherein said first comparison result is subtracted from the EPR measurement of another non-treated blood subsample thereby obtaining a second comparison result which is an EPR spectrum; and determining the NO quantity in said collected whole blood sample from said second comparison result using a hyperfine component.
  • said hyperfine component is hyperfine component of the triplet hyperfine structure of EPR
  • a whole blood sample is collected directly into a gas-tight vacuum tube and immediately centrifuged.
  • the direct collection of the whole blood sample into a gas-tight vacuum tube is an anaerobic approach that prevents the dissociation of the in vivo formed Hb-NO complex.
  • the gas-tight vacuum tube contains an anticoagulant such as heparin or sodium citrate, preferably Ethylene diamine tetra acetic acid (EDTA).
  • an anticoagulant such as heparin or sodium citrate, preferably Ethylene diamine tetra acetic acid (EDTA).
  • the whole blood sample is collected from an animal blood or from human blood.
  • the blood sample is venous blood, more preferably collected from the median antebrachial vein of a human.
  • centrifugation of the collected blood sample is carried out at a rotation speed comprised between 550 g and 1500 g, preferably between 650 g and 850 g, more preferably between 700 g and 800 g.
  • Centrifugation time is comprised between 20 min and 2 min, preferably between 17 min and 5 min, more preferably between 15 min and 7 min.
  • Centrifugation temperature is comprised between 15°C and 1°C, preferably between 10°C and 2°C, more preferably between 5°C and 3°C.
  • the plasma is discarded after centrifugation of the collected whole blood sample and the erythrocytes or red blood cells (RBCs) are kept for further use.
  • the obtained erythrocytes are divided in at least two blood subsamples. These blood subsamples are collected from the bottom of the gas-tight vacuum tube using a Pasteur glass pipette. Collection of blood subsamples from the bottom of the gas-tight vacuum tube ensures the presence of erythrocytes in the collected blood subsamples. At least 0.3 ml of the obtained erythrocytes are collected in each blood subsample.
  • a first erythrocytes subsample is immediately frozen at about 77°K (-196 °C) in liquid nitrogen.
  • This first RBCs subsample contains the in vivo formed Hb-NO.
  • a second and a third blood subsample are allowed to settle at a temperature comprised between 5 °C and 15 °C, preferably between 7 °C and 13 °C, more preferably between 8 °C and 11 °C, even more preferably about 10 °C.
  • the said second and third blood subsamples are allowed to settle for a time comprised between 15 min and 60 min, preferably between 20 min and 45 min, more preferably between 25 min and 35 min. Allowing said second and third blood subsamples to settle will lead to the dissociation of the Hb-NO complex in said subsamples.
  • a chemical compound is added to the second blood subsample to reduce at least partly the free radicals present in said second blood subsample.
  • the chemical compound is an antioxidant, such as but not limited to ascorbic acid.
  • the antioxidant is an ascorbic acid solution with a final molar concentration comprised between 1 mM and 20 mM, preferably between 5 mM and 15 mM, more preferably between 5 mM and 12 mM, even more preferably around 10 mM.
  • the antioxidant is added to the blood subsample in volume proportion 1 : 5, 1 : 6, 1 : 7, 1 :8, 1 :9, 1 : 10, 1 : 11, 1 : 12, 1 : 13, 1 : 14 or 1 : 15.
  • the antioxidant is added to the blood subsample in volume proportion of 1 : 10.
  • the second and the third blood subsamples are again allowed to settle at a temperature comprised between 5 °C and 15 °C, preferably between 7 °C and 13 °C, more preferably between 8 °C and 11 °C, even more preferably about 10 °C.
  • said second and third blood subsamples are allowed to settle for a time comprised between 30 min and 5 min, preferably between 25 min and 10 min, more preferably between 20 min and 10 min, even more preferably for about 15 min.
  • the second and the third blood subsamples are frozen at about 77°K in liquid nitrogen.
  • EPR measurements are performed for all blood subsamples.
  • a volume taken from the RBC's of each blood subsample is placed by a Pasteur glass pipette into a 1ml plastic syringe which is cut off at the tip, the opened end of the syringe is closed with small sheet of parafilm and the subsample is immediately frozen in liquid nitrogen (-196°C) before EPR measurements.
  • Said volume is comprised between 0.1 ml and 0.5 ml, preferably between 0.2 ml and 0.4 ml, more preferably about 0.3 ml.
  • each blood subsample is pushed out, while still frozen, from the syringe.
  • the frozen sample is then transferred into a finger Dewar flask filled with liquid nitrogen.
  • an EPR-silent material such as but not limited to a filter paper or cotton was inserted into the gap between the frozen sample and the wall of the finger Dewar flask.
  • the EPR measurements are carried out using an X-band EMX (Bruker Instruments Inc.) and a quartz finger Dewar under the following instrumental parameters: microwave frequency, ⁇ 9.35 GHz; modulation frequency, 100 KHz; centre field 3270 G; diapason 450 G; microwave power, 20 mW; modulation amplitude, 7G, time constant 40.9 ms, gain 6.32xl0 4 ; 10 scans, temperature 77K, and with control of quality factor Q.
  • EPR measurements are carried out using an X-band MiniScope MS400 (Magnettech GmbH .) and a quartz finger Dewar under the following instrumental parameters: microwave frequency, ⁇ 9.4 GHz; modulation frequency, 100 KHz; centre field 3298 G; diapason 449 G; microwave power, 20 mW; modulation amplitude, 7G, gain 500; 10 scans, temperature 77K.
  • the obtained EPR spectra of the different blood subsamples are used in at least two subtractions.
  • the EPR spectrum of the second chemically treated blood subsample is subtracted from the EPR spectrum of the third non-chemically treated blood subsample using computer software. This first subtraction will result in an EPR model spectrum of free radicals specific to the subject from which the whole blood sample was collected.
  • the obtained EPR model spectrum of free radicals (with peak-to-peak width about 18 G) is subtracted from the EPR spectrum of the first non-treated blood subsample (containing the in vivo formed Hb-NO), using proportional coefficient obtained as a peak-to-peak amplitude ratio within the obtained model EPR spectrum of free radicals and the peak-to-peak amplitude of free radical EPR spectrum from first untreated subsample frozen immediately after centrifugation.
  • the invention provides a kit for carrying out the NO measurement method.
  • the kit contains at least a winged infusion set with needle (21Gx3/4") and a gas-tight vacuum tube with adaptor for blood sampling, a chemical solution, at least 4 Pasteur glass pipettes, at least 3 plastic syringes (1 ml) cut off at the end for subsample freezing, at least 3 small sheets of parafilm to close open end of syringe after filling and a protocol description.
  • the gas-tight vacuum tube provided in the kit contains an anticoagulant such as heparin or sodium citrate, preferably EDTA.
  • the use of the method and kit according to the invention provides an easy approach for an accurate measurement of the NO levels in human blood.
  • the described method can be used for the medical diagnostic of vascular diseases. It will improve the diagnostic as it allows the detection of endothelial dysfunction at a relatively early stage.
  • the first subsample was frozen immediately at 77°K in liquid nitrogen.
  • Ascorbic acid (final concentration 10 mM) was added to second blood subsample after 30 minutes for free radical reduction. Ascorbic acid was added to the blood subsample in a volume proportion 1 : 10.
  • the instrumental parameters for EPR measurements by X-band EMX were as follows: microwave frequency, ⁇ 9.33 GHz; modulation frequency, 100 KHz; centre field 3270 G; diapason 450 G; microwave power, 20 mW; modulation amplitude, 7G, time constant 40.9 ms, gain 6.32xl0 4 ; 10 scans. Three EPR spectra were obtained.
  • FIG. 1 Model signal of free radicals (a) obtained after first subtraction.
  • the highest EPR spectrum (c) was obtained from the third non-chemically treated blood subsample.
  • the EPR spectrum in the middle (b) was obtained from the second chemically treated blood subsample.
  • the lowest EPR spectrum (a) is the model spectrum of free radicals specific to the patient from which the whole blood sample was collected.
  • the final obtained spectrum was used for the quantification of Hb-NO using high- field hyperfine (hf) structure of 5-coordinated nitrosylated a-Hb as a peak-to-peak amplitude of hf component of the triplet hyperfine structure of EPR spectrum in diapason 3299-3307 Gauss at microwave frequency ⁇ 9.34 GHz.
  • hf high- field hyperfine
  • the spectrum observed after addition of NO donor to isolated RBCs in vitro was used as an etalon control as shown in FIG. 2 and FIG.3, i and i' respectively.
  • FIG. 2 Example of double subtraction (volunteer 1) :
  • B. EPR spectrum of RBCs of healthy human volunteer 1 (k), presented as a high field component, after final subtraction of the model EPR spectrum of free radicals (d) divided by the proportional coefficient (K 2.4 in this experiment) from the EPR spectrum of the first subsample (h) frozen immediately after sampling.
  • volume (volunteer 1). Signal of 5-coordinated nitrosylated a-Hb in RBCs incubated with nitric oxide donor in vitro (spectrum Hb- NO) is presented as an etalon in a 1 : 6 scale for comparison (i).
  • FIG. 3 Example of double subtraction (volunteer 2) :
  • B. EPR spectrum of RBCs of healthy human volunteer 2 (k'), presented as a high field component, after final subtraction of the model EPR spectrum of free radicals (d') divided by the proportional coefficient (K 0.52 in this experiment) from the EPR spectrum of first subsample (h') frozen immediately after sampling.
  • volumenteer 2 Signal of 5- coordinated nitrosylated a-Hb in RBCs incubated with nitric oxide donor in vitro (spectrum Hb-NO) is presented as an etalon in a 1 : 6 scale for comparison (i').
  • RHI reactive hyperaemia response
  • PAT peripheral arterial tone
  • RHI reactive hyperaemia response
  • Quantitation of the Hb-NO level was perfomed using the calibration curve obtained from the EPR signals of Hb-NO formed in RBCs treated with known concentration of nitrite in presence of Na 2 S 2 0 4 in anaerobic condition (37°C; 1% of 0 2 ; RUSKINN workstation INVIVO 2 400).
  • FRHI Framingham index of reactive hyperaemia response
  • the Framingham reactive hyperemia index (FRHI) is defined as the natural logarithm of the ratio of mean post-deflation signal (in the 90 to 120-second post-deflation interval) to the baseline signal in hyperemic finger normalized by the same ratio in the contra-lateral finger.
  • the calibration curve used for quantification of amount of Hb-NO, formed in erythrocytes is shown in Fig. 5.
  • the calibration curve (A) was obtained from the EPR signals of Hb-NO (B) formed in intact RBCs treated with known concentration of nitrite in presence of Na 2 S 2 0 4 (20 mM) in anaerobic condition (1% of 0 2 ; RUSKINN workstation INVIVO 2 400; 37°C).
  • nitric oxide formed in equimolar ratio from nitrite after reduction by Na 2 S 2 0 4 , proportionally interacts with deoxy-haemoglobin.
  • the EPR signals were standardized by comparison of the spectrum intensity after double integration with intensity of known stable standard, a buffer solution of Cu(EDTA) frozen in 30% of glycerol at concentration 50 and 100 ⁇ .
  • FIG. 6 shows individual model EPR spectrum of free radicals (a) in RBCs obtained after inverse subtraction of the EPR spectrum of the non-treated subsample (c) from the EPR spectrum of third subsample treated with ascorbic acid (10 mM) (b) in RBCs with prooxidant profile.
  • N-acetyl cysteine N-acetyl cysteine
  • a-tocopherol 4-10 mM
  • N-acetyl cysteine 10 mM
  • Fig 7 A shows individual model EPR spectrum of free radicals (a) in RBCs obtained after subtraction of the EPR spectrum (b) of the subsample treated with N-acetyl cysteine (NAC, 10 mM), from the EPR spectrum of third non-treated subsample (c).
  • Fig 7 B shows individual model EPR spectrum of free radicals (a) in RBCs obtained after subtraction of the EPR spectrum (b) of the subsample treated with a- tocopherol (4 mM) from the EPR spectrum of third non-treated subsample (c).
  • Fig. 7- IA shows individual model EPR spectra of free radicals (a and a') in RBCs obtained after subtraction of the EPR spectrum of the subsample treated with different N-acetyl cysteine (NAC) concentrations, 5 mM for subsample (b) and 10 mM for subsample (b'), from the EPR spectrum of third non-treated subsample (c).
  • Fig. 7-IB shows comparative model EPR spectra of free radicals obtained by using a subsample treatment with different concentrations of ascorbic acid (2 and 10 mM).
  • Fig. 7-IC shows a comparison of different antioxidant effect.
  • the model EPR spectra of free radicals in RBCs obtained from a single volunteer after subtraction of the EPR spectrum of the subsample treated with different antioxidants from the same EPR spectrum of third non-treated subsample.
  • the effect of a-tocopherol 4 mM is shown in a; effect of Trolox which is a water-soluble analog of a-tocopherol 1.7 mM is shown in b and effect of N-acetyl cysteine 10 mM is shown in c.
  • All model spectra, being divided by correspondent proportional scale factor, were used for subtraction. Proportional scale factors were k 1.33 (a); 0.75 (b); 0.96 (c) related to intensity of free radical signal in first non-treated subsample.
  • Fig 8-1 shows the linear regression analysis between the Framingham index of reactive hyperaemia response (FRHI), calculated automatically by the EndoPat2000 software, and the level of Hb-NO, quantified after subtraction of model free radical signal obtained with antioxidant treatment of RBCs in vitro as described above.
  • FRHI Framingham index of reactive hyperaemia response
  • Test of NO formation in circulation determined by the accumulation of Hb-NO in RBCs obtained from a volunteer after in vivo treatment with glyceryl trinitrate (GTN).
  • RBCs were isolated as described previously from blood of a volunteer 2 minutes before and 2 minutes after acute GTN administration (sublingual GTN spray, 2 doses, 0,4 mg/dose). Hb-NO level was increased to nearly twice the basal level after 2 minutes of the acute in vivo GTN administration.
  • Typical EPR spectra of RBCs, frozen after incubation under low 0 2 level with the NO-donor, Dea-NONOate (l,l-Diethyl-2-hydroxy-2-nitroso- hydrazine, 50 ⁇ /L; gain 3.1xl0 4 ) are shown at the bottom of the figure (Hb- NO model) for comparison.
  • Peak-to-peak amplitude of hf component of the Hb-NO triplet structure attributed to 5-coordinated nitrosylated a-Hb was used for quantitation. Spectra are recorded using Bruker spectrometer (X-band, microwave frequency ⁇ 9.35 GHz) with the following setting : modulation frequency, 100 kHz; microwave power (MP), 20 mW; modulation amplitude (MA), 7 mT; 10 scans, 77K.
  • Test of NO formation in circulation determined by the accumulation of Hb-NO in RBCs, obtained from volunteer after five-minute exercise (stairs climbing).
  • Spectra are recorded using Bruker spectrometer (X-band, microwave frequency ⁇ 9.35 GHz) with setting : modulation frequency, 100 kHz; microwave power (MP), 20 mW; modulation amplitude (MA), 7 mT; 10 scans, 77K.
  • Test of NO formation in circulation determined as the accumulation of Hb-NO in RBCs, obtained from patient using an X-band MiniScope MS400 (Magnettech GmbH .) and a quartz finger Dewar et the temperature 77K.
  • Spectra are recorded using EPR spectrometer MiniScope MS400 (Magnettech GmbH ., X-band) microwave frequency ⁇ 9.4 GHz with setting : modulation frequency, 100 kHz; microwave power (MP), 20 mW; modulation amplitude (MA), 7 mT; 10 scans, 77K.
  • EPR spectrometer MiniScope MS400 Magnnettech GmbH ., X-band microwave frequency ⁇ 9.4 GHz with setting : modulation frequency, 100 kHz; microwave power (MP), 20 mW; modulation amplitude (MA), 7 mT; 10 scans, 77K.

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Abstract

La présente invention concerne un procédé de quantification du monoxyde d'azote NO dans un échantillon sanguin comprenant les étapes de prélèvement d'un échantillon sanguin, de division dudit échantillon sanguin prélevé en trois sous-échantillons sanguins, de traitement chimique d'un sous-échantillon sanguin avec un antioxydant pour au moins réduire en partie les radicaux libres dudit sous-échantillon sanguin, d'exécution d'une mesure de résonance paramagnétique électronique (RPE) desdits sous-échantillons sanguins, d'exécution d'une première comparaison des mesures de RPE desdits sous-échantillons sanguins traités chimiquement avec la mesure de RPE d'un sous-échantillon sanguin non traité chimiquement, permettant ainsi d'obtenir un premier résultat de comparaison, et d'exécution d'une deuxième comparaison de ce résultat avec une mesure de RPE du sous-échantillon sanguin restant afin d'éliminer les signaux de bruit de fond.
PCT/EP2012/076904 2011-12-27 2012-12-26 Procédé de mesure du monoxyde d'azote dans le sang WO2013098294A1 (fr)

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WO2015009970A1 (fr) 2013-07-18 2015-01-22 Erythron Llc Mesures spectroscopiques avec un détecteur de réseaux parallèles
WO2015131151A2 (fr) 2014-02-28 2015-09-03 Erythron, Llc Méthode et appareil de détermination de marqueurs de santé par analyse d'un échantillon de sang
WO2016168090A1 (fr) 2015-04-14 2016-10-20 Nueon, Inc. Méthode et appareil pour déterminer des marqueurs de santé par analyse de sang
WO2017165403A1 (fr) 2016-03-21 2017-09-28 Nueon Inc. Procédés et appareil de spectrométrie à maillage poreux
WO2018085699A1 (fr) 2016-11-04 2018-05-11 Nueon Inc. Lancette et analyseur de sang combinés
CN111989095A (zh) 2018-04-16 2020-11-24 上海岸阔医药科技有限公司 预防或治疗肿瘤疗法副作用的方法

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US10598652B2 (en) 2015-04-09 2020-03-24 The Regents Of The University Of California Methods of diagnosing post-operative complications using serum nitric oxide levels
CN108459043A (zh) * 2018-03-05 2018-08-28 大连工业大学 检测海藻提取物加工残渣对饼干脂质氧化抑制效果的方法

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